The corticotropin releasing factor (CRF) type 1α receptor, a member of the G protein-coupled receptor (GPCR) subfamily B, is involved in the aetiology of anxiety and depressive disorders. In the present study, we examined the internalization and trafficking of the CRF1α receptor in both human embryonic kidney (HEK)293 cells and primary cortical neurons. We found that CRF1α receptor activation leads to the selective recruitment of β-arrestin2 in both HEK293 cells and neurons. We observed distinct distribution patterns of CRF1α receptor and β-arrestin2 in HEK293 cells and cortical neurons. In HEK293 cells, β-arrestin2-green fluorescent protein (GFP) co-localized with CRF1α receptor in vesicles at the plasma membrane but was dissociated from the receptor in endosomes. In contrast, in primary cortical neurons, β-arrestin2 and CRF1α receptor were internalized in distinct endocytic vesicles. By bioluminescence resonance energy transfer, we demonstrated that β-arrestin2 association with CRF1α receptor was increased in cells transfected with G protein-coupled receptor kinase (GRK)3 and GRK6 and decreased in cells transfected with GRK2 and GRK5. In both HEK293 cells and cortical neurons, internalized CRF1α receptor transited from Rab5-positive early endosomes to Rab4-positive recycling endosomes and was not targeted to lysosomes. However, CRF1α receptor resensitization was blocked by the overexpression of wild-type, but not dominant-negative, Rab5 and Rab4 GTPases. Taken together, our results suggest that β-arrestin trafficking differs between HEK293 cells and neurons, and that CRF1α receptor resensitization is regulated in an atypical manner by Rab GTPases.